PURIFICATION AND CHARACTERIZATION OF THE HUMAN KDEL RECEPTOR

Retention of soluble endoplasmic reticulum (ER) proteins is ensured by their continuous retrieval from subsequent compartments in the secret ory pathway. Soluble ER proteins which escape to the Golgi apparatus b ind to the KDEL receptor, a seven-transmembrane receptor, and are then returned to the endoplasmic reticulum, We have overexpressed the huma n KDEL receptor in insect cells using the baculovirus system. Infected cells accumulate large amounts of functional receptor as judged by a ligand binding assay. A hexahistidine-tagged version of the receptor c ould be purified in a single step to near homogeneity with high yield. After reconstitution of purified KDEL receptor into liposomes, a simi lar affinity and pH dependence for the binding of KDEL peptides was ob served compared to the receptor in its natural environment, indicating that purified KDEL receptor is sufficient for specific and pH-sensiti ve binding of KDEL ligands. Determination of the receptor affinity in different lipid environments revealed that the receptor affinity is on ly slightly influenced by its lipid environment, suggesting that regul ation of the receptor affinity by its surrounding lipids does not play a crucial role for the sorting of KDEL proteins.